With obesity now affecting one in five children in the U.S. and gestational diabetes mellitus (GDM) increasing among all racial and ethnic groups, it is becoming increasingly important to understand how events that happen early in life, even before birth, can alter the obesity trajectory of individuals. The fetal over-nutrition hypothesis posits hat intrauterine environment of diabetic and obese pregnancies may permanently alter the offspring's long- term risk for obesity and metabolic diseases, through developmental programming. This risk is hypothesized to operate through permanent changes in cell and tissue structure and function induced by changes in expression of target genes. Epigenetic modifications are mediators of the effect of the environment on gene expression and are likely to be a crucial mechanism involved in these processes. The EPOCH (Exploring Perinatal Outcomes in Relationship CHildren) study is a historical prospective cohort that enrolled children aged 10.5 on average (first EPOCH visit T1 when maternal and offspring peripheral blood was collected) who were exposed or not exposed to maternal GDM during the intrauterine life. EPOCH offspring are now transitioning through puberty and will be followed for another five years (EPOCH renewal recently funded; 2R01 DK068001-6) with a second EPOCH visit (T2) occurring on average at 16.5 years and a planned collection of peripheral blood DNA and RNA for epigenetic and transcriptional studies. Given the established role of epigenetics in mediating the effects of exposure on gene expression, the overarching hypothesis of this proposal is that exposure to maternal GDM in utero will be associated with changes in DNA methylation patterns of key genes/pathways in the offspring and that these epigenetic changes will mediate the association between in utero exposure and childhood/adolescence adiposity-related outcomes. We will test this hypothesis through the three specific aims outlined in Figure 1 in 85 exposed and 255 unexposed children. Briefly, we intend to identify DNA methylation changes that are associated with in utero GDM exposure (Aim 1), followed by identification of epigenetic marks that are causal and mediate adiposity-related outcomes (Aim 2), and to identify DNA methylation marks (and associated gene expression changes) that persist into adolescence (Aim 3).